Prevalence of Listeria monocytogenes in food samples from retail shops and street vendor stalls in Pretoria and the evaluation of bacterial probiotics as potential control measure By Brighton Ncube Submitted in partial fulfillment of the requirements for the degree of Magister Scientiae In the Department of Biochemistry, Genetics and Microbiology Faculty of Natural and Agricultural Sciences University of Pretoria Pretoria South Africa Supervisor: Prof MS Thantsha Co Supervisor: Dr MG Mathipa June 2020 © University of Pretoria DECLARATION I declare that the dissertation ‘Prevalence of Listeria monocytogenes in food samples from retail shops and street vendor stalls in Pretoria and the evaluation of bacterial probiotics as potential control’ which I hereby submit for the degree of Magister Scientae at the University of Pretoria, is my own work and has not been previously submitted by me for a degree at this or any other tertiary institution. Signature: Date: 30 June 2020 ii © University of Pretoria Table of Contents Acknowledgements……………………………………………………………………………..... vii Conference contributions………………………………………………………………………… viii List of abbreviations………………………………………………………………………………. ix List of tables……………………………………………………………………………..………… xii List of figures………………………………………………………………………….…………… xiii Dedication…………………………………………………………………………………………. xiv Summary…………………………………………………………………………….…………….. 1 Introduction……………………………………………………………………………………....... 6 CHAPTER 1: LITERATURE REVIEW 1.1 L. monocytogenes history and taxonomy……………………………………….………… 14 1.2 Sources of contamination…………………………………………………………………… 16 1.3 Foods implicated in L. monocytogenes outbreaks………………………………............ 17 1.3.1 Dairy…………………………………………………………………………………. 17 1.3.2 Poultry……………………………………………………………………………….. 18 1.3.3 Meat and Fish……………………………………………………...….……………. 18 1.3.4 Vegetables…………………………………………………………………............. 18 1.3.5 Ready to eat food…………………………….….…………………………………. 19 1.4 Control of L. monocytogenes………………………………………………………………. 19 1.5 L. monocytogenes infection …..…………………………………………….….………….. 21 1.6 Listeriosis outbreaks ……...……………………………………………………………….. 25 1.7 Antibiotics and listeriosis…..……………………………………………………………… 27 1.8 Probiotics……………………………………………………………...…………………….. 29 1.8.1 What are they? ………………………………………………………….……....... 29 1.8.2 History of probiotics……………………………………………………………….. 30 1.8.3 Characteristics of probiotics………………………………………………………. 30 1.8.4 Probiotics in food production…………………….……………………....……….. 32 1.8.5 Gastrointestinal activity………………………………………………...…............. 32 iii © University of Pretoria 1.8.6 Antimicrobial activity……………………………...………………………………... 33 1.8.7 Strengthening epithelial barrier function……………....…….………….............. 36 1.8.8 Modulating the immune system response……………...………........................ 36 1.9 Probiotics and disease………………………………………………………..................... 37 1.9.1 Probiotics and food allergy …………………………………………….....…….... 37 1.9.2 Antibiotic associated diarrhoea…..……………………………………............. 38 1.9.3 Probiotics and Necrotizing enterocolitis……………………….…...................... 38 1.9.4 Inflammatory bowel disease…………..…………………………………...……... 38 1.10 L monocytogenes and probiotics …………….………………………………………....... 39 1.10.1 Production of antimicrobial substances…………………………………………. 39 1.10.2 Inhibiting adhesion……………………………………………………….….......... 39 1.10.3 Mouse models…………………………………………………………….….......... 40 1.11 Significance of study…….….…………………….………………………...........………... 40 1.12 References ……………..….………………………………………………..………............ 42 CHAPTER 2: COMPARATIVE ANALYSIS OF THE PREVALENCE OF LISTERIA MONOCYTOGENES , SALMONELLA SPP AND CAMPYLOBACTER SPP IN RETAILS STORES AND STREET VENDORS STALLS IN PRETORIA 2.1 Abstract……………………………………………………………………………................ 85 2.2 Introduction…………………………………………………………………………..……… 86 2.3 Materials and Methods……………………………………………………………………… 87 2.3.1 Collection of samples……………………………………………………...………. 87 2.3.2 Microbiological analysis …………………………………………………………... 88 2.3.2.1 Isolation of Listeria ……………………………….................................. 88 2.3.2.2 Isolation of Salmonella ……………………………................................ 89 2.3.2.3 Isolation of Campylobacter ………………………………...……………. 89 2.3.2.4 Gram staining …………………………………………………....……….. 89 2.3.3 DNA extraction …………………………………………………........................... 90 iv © University of Pretoria 2.3.4 Amplification of 16S rRNA gene using PCR …………………………………… 90 2.3.5 Cleaning of PCR products ……........................................................................ 90 2.3.6 Sequencing of amplicons ……………..……………………............................... 91 2.3.7 Amplification of inlA using PCR ……………….………………….…….…......... 91 2.3.8 Antibiotic susceptibility tests …............……..………………………….............. 92 2.4 Results ……………………………………….…………………………….…………......... 93 2.4.1 Presumptive isolates using plating ……..……………………………….……….. 93 2.4.2 DNA extraction……………………………………….…………………………….. 93 2.4.3 16SrRNA gene amplification …………………………….…………….…………. 94 2.4.3.1: Listeria …………………………………………………………………….. 94 2.4.3.2 Salmonella ………..……………………….……………………….……… 94 2.4.3.3 Campylobacter …………………………..……………………................. 95 2.4.4 Sequencing and BLAST analysis …………….................................................. 96 2.4.5 Amplification of the inlA gene from suspected L. monocytogenes isolates…… 97 2.4.6 Prevalence of L. monocytogenes ……………………………………….............. 98 2.4.7 Antibiotic susceptibility …………………………………………………..………... 98 2.5 Discussion ………………………………………………………………………..………… 99 2.6 Conclusion ……………………………………………………………..…………………... 102 2.7 References …………………………………………………………………………………. 103 CHAPTER 3: EVALUATION OF DIFFERENT PROBIOTIC STRAINS AS A CONTROL MEASURE AGAINST LISTERIA MONOCYTOGENES 3.1 Abstract………………………………………………………………………….…..…….... 115 3.2 Introduction ……………………………………………………………………..………….. 116 3.3 Materials and Methods …………………………………………………………............... 118 3.3.1 Bacterial cultures………………………………………………………..…............ 118 3.3.2 Spot inoculation tests ……………………………………………………............. 118 3.3.3 Preparation of cell free supernatants (CFS) of probiotics ………..…………… 119 3.3.4 Well diffusion assay ……..…………………………………………...………….... 119 v © University of Pretoria 3.3.5 L. monocytogenes growth in avocado and cucumber…………………………... 120 3.3.6 Growth of L. monocytogenes in food samples in presence of probiotics…… 120 3.3.7 Growth of L. monocytogenes in food samples in presence of probiotic CFS…. 121 3.3.8 Growth of L. monocytogenes on food sample in presence of probiotic cocktails…………………………………………………………………..……….... 121 3.3.9 Statistical analysis ……………………………………………………………….... 122 3.4 Results ………………………………………………………………………………........... 122 3.4.1 Spot inoculation test ……………………………………………………................ 122 3.4.2 Well diffusion assay ……………………………………………………….……… 123 3.4.3 Probiotics against L. monocytogenes in food samples ……………………...... 123 3.4.4 Probiotic cell free supernatants against L. monocytogenes in food sample….. 124 3.4.5 Probiotic cocktails against L. monocytogenes in food sample………………....... 126 3.5 Discussion ……………………………………………………………………………......... 127 3.6 Conclusion ………………………………………………………….................................. 129 3.7 References …………………………………………………………................................. 130 CHAPTER 4: GENERAL CONCLUSIONS AND RECOMMENDATIONS General conclusions………………………………………………………………………............ 139 Recommendations…………………………………………………………………………........... 141 vi © University of Pretoria ACKNOWLEDGEMENTS First and foremost, all praises due to JEHOVAH for such an illustrious platform. It is only in His presence and according to his mercy that we can do whatever we do as we walk this earth. To my mother, Pretty Ncube, I have no words. If these words could cry, my whole dissertation would soak. My family, whose patience and love seems to flow from an ever abundant source. My love, April B Evans, who has been by my side through the best and worst moments of this undertaking. To my friends far and wide for being pillars to lean on when I buckled. To my supervisor, Prof MS Thantsha, thank you for taking me under your wing and allowing me to gain from the waters of your wisdom. Your knowledge and patience with me I will take to heart and to skill for the rest of my days. To my co supervisor Dr Moloko Mathipa whose perseverance and kind heart made for a great mentor without whom all this would not be possible. To my lab mates, past and present, who have been part of my daily highs and lows, thanks for taking part in my journey. As rocky as it was, you smoothened the curve. Lastly, thank you to the National Research Foundation (NRF) and University of Pretoria for providing me with funding. vii © University of Pretoria Conference contributions Ncube, B. N. Mathipa M.G. Thantsha M.S “PREVALENCE OF L. MONOCYTOGENES IN RETAIL SHOPS AND STREET VENDORS AROUND PRETORIA AND EVALUAITION OF PROBIOTICS AS A CONTROL MEASURE” 23rd Biennial International Congress and Exhibition- SAAFoST 2019. Birchwood Hotel and Conference Centre, Johannesburg. 1-4 September 2019. (Poster presentation). viii © University of Pretoria LIST OF ABBREVIATIONS AAD : Antibiotic associated diarrhoea actA : Actin A ANOVA : Analysis of Variance avoS : avocado isolate BA-LA : Bifidobacterium animalis – Lactobacillus acidophilus cocktail BA-PA : Bifidobacterium animalis – Pediococcus acidilacti cocktail BHI : Brain Heart Infusion BLAST : Basic Local Alignment Search Tool BMEC : Brain Macrovascular Endothelial Cells Bp : Base pairs CBD : Central Busines District CDC : Centre for Disease Control